PRELI and oxidative stress down-regulate STAT6 and activate calpain, a known STAT6 protease. (A) Knockdown of PRELI leads to increase of STAT6 at the protein level. Nucleofected CD4⁺ cells were activated for 24 hours and subsequently harvested for Taqman RT-PCR analysis and Western blotting. Protein bands were quantified with a microcomputer imaging device (MCID) and normalized against β-actin. Bars represent the average fold changes plus or minus SEM, calculated from independent experiments (STAT6 protein, n = 10; STAT6 mRNA, n = 4). (B) PRELI does not influence the tyrosine (Y641) phosphorylation of STAT6. CD4⁺ cells nucleofected with pIRES-H2K^k or the PRELI overexpression vector were activated and cultured in the presence of IL-4 for 1 hour and subsequently harvested for Western blotting. Protein bands were quantified and normalized, as in panel A. Representative data from 5 independent experiments are shown. (C) STAT6 is down-regulated at the protein level by oxidative stress in human primary Th cells. Cord blood CD4⁺ cells were activated for 20 hours and subsequently exposed to increasing concentrations of H₂O₂ for 4 hours. The protein and mRNA levels of STAT6 were analyzed by Western blotting and Taqman RT-PCR analysis. A portion of the cells was stained with DHR123, to assess ROS production, and analyzed by flow cytometry. Representative data from 4 independent experiments are shown. (D) Neutralization of intracellular ROS increases the level of STAT6. CD4⁺ cells were activated and cultured with or without 1.25 mM NAC for 24 hours, after which the cells were harvested for Western blotting. STAT6 protein bands were quantified and normalized as in panel A. Representative data from 6 independent experiments are shown. (E) Oxidative stress induces calpain activation in human Th cells. Exposure of the cells to H₂O₂ was performed as in panel C (black arrow, inactive procalpain [80 kDa]; gray arrow, active calpain [75 kDa]). The active and inactive calpain bands were quantified with an MCID and the active versus inactive calpain ratios were calculated in each sample. Subsequently, the active/inactive calpain ratio of the control (untreated) sample was set as 1 and the fold changes of the ratios of the other samples were calculated in relation to the control sample. Bars represent the mean values plus or minus SEM of the fold changes of different replicates. Representative data from 2 experiments are shown. (F) STAT6 is up-regulated in response to calpain inhibition. CD4⁺ cells were activated and cultured for 24 hours with or without calpastatin peptide (5 μM) and subsequently harvested for Western blotting. STAT6 protein bands were quantified with an MCID and normalized against β-actin. Vertical lines have been inserted to indicate a repositioned gel lane. The relative ratios of active versus inactive calpain were determined as described in panel E. Representative data from 5 independent experiments are shown. (G) Overexpression of PRELI in Hek293 cells induces activation of calpain. Hek293 cells transfected with a control vector (empty pFlag) or pFlag-PRELI were harvested for Western blotting 24 hours after transfection. The relative ratios of active versus inactive calpain were determined as described in panel E. Data are representative of 2 independent experiments. (A,B,D,F) Statistical significances were determined using the 2-tailed paired t test. OE indicates overexpression.